Methods for determining responsiveness to mek/erk inhibitors

ABSTRACT

A method for predicting the responsiveness of a cancer cell to an MEK inhibitor, comprising detecting the presence of at least one mutation in one or more genes selected from the group consisting of ADAM12, COL14A1, TNN, and TP53, in the cancer cell, by contacting a nucleic acid sample derived from the cancer cell with at least one oligonucleotide which allows specific detection of the mutation; wherein presence of mutation in ADAM12, COL14A1, TNN, TP53 and/or any combination thereof is indicative of decreased responsiveness of the cancer cell to the ERK inhibitor.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional application of U.S. Ser. No. 15/301,379, which is the national phase stage of PCT/CN2015/075884 filed on Apr. 3, 2015, which claims the priority to Chinese patent application no. 201410135569.9, filed Apr. 4, 2014, the disclosure of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention generally relates to methods for determining the responsiveness of a subject to treatment with a MEK or ERK inhibitor.

BACKGROUND OF THE INVENTION

The Ras-Raf-MEK-ERK signaling cascade (MEK/ERK pathway) is one of key pro-proliferation and pro-survival pathways. Mutations in the MEK/ERK pathway have been found to lead to uncontrolled growth in many cancers (e.g., melanoma). Compounds that inhibit steps in the MEP/ERK pathway have been used to treat cancer. However, some patients that harbor mutations in MEK/ERK pathway show resistance to MEK or ERK inhibitors.

There is a need for an effective means of determining which patients having mutations in MEK/ERK pathway will resist to treatment of MEK or ERK inhibitors and for incorporating such determination into effective treatment.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present disclosure provides a method for predicting the responsiveness of a cancer cell to an MEK inhibitor. In certain embodiments, the method comprises detecting the presence of at least one mutation in one or more genes selected from the group consisting of ADAM12, COL14A1, TNN, and TP53, in the cancer cell, by contacting a nucleic acid sample derived from the cancer cell with at least one oligonucleotide which allows specific detection of the mutation; wherein presence of mutation in ADAM12, COL14A1, TNN, TP53 and/or any combination thereof is indicative of decreased responsiveness of the cancer cell to the MEK inhibitor.

In certain embodiments, the cancer cell is derived from a cancer patient.

In certain embodiments, the MEK inhibitor is Trametinib.

In certain embodiments, the mutation in ADAM12 is selected from the group consisting of mutation Q650K, R240L, C440Y, Q228E, H247D, M322I, T97fs, P168L and G308E in ADAM12; the mutation in COL14A1 is selected from the group consisting of mutation R178W, L713 splice, Q1272K, L479I, L1295F, E1024K, P1467S, G737R, K1023T, G966C, S1512fs in COL14A1; the mutation in TNN is selected from the group consisting of mutation V353M, Y296S, A733P, D707Y, D471Y, P1010T, S71L, D457Y, P1155L, R476C, Q872H, Q261L, D798Y, C1237*, D67N and T823S in TNN; the mutation in TP53 is selected from the group consisting of mutation Q331R, C135fs, E285K, V274F, Y220C, P250L, R175H, R248Q, R280K, R248L, C176Y, A307_splice, R273L, R158L, A138fs, H193R, A159D, C277F, R248W, Y220C, V274F, R196*, E224_splice, K164*, M246I, A159V, S241F, C242R, S261_splice, E339* in TP53.

In certain embodiments, the detecting step comprises amplifying at least a portion of the gene with the oligonucleotide as primer, and detecting the amplification product and thereby determining the presence of the mutation in the gene.

In certain embodiments, the detecting step comprises contacting the nucleic acid sample with the oligonucleotide which specifically hybridizes to the mutation of the gene to form a complex, and detecting the formation of the complex and thereby determining the presence of the mutation in the gene.

In anther aspect, the present disclosure provides a method of identifying a likely responder or a likely non-responder to an MEK inhibitor, comprising detecting the presence of at least one mutation in one or more genes selected from the group consisting of ADAM12, COL14A1, TNN, and TP53, in a sample from the patient, by contacting the sample with at least one oligonucleotide which allows specific detection of the mutation; identifying the patient as a likely non-responder to the MEK inhibitor if at least one mutation in in ADAM12, COL14A1, TNN, TP53 and/or any combination thereof is detected in the sample.

In certain embodiments, the MEK inhibitor is Trametinib.

In certain embodiments, the mutation in ADAM12 is selected from the group consisting of mutation Q650K, R240L, C440Y, Q228E, H247D, M322I, T97fs, P168L and G308E in ADAM12; the mutation in COL14A1 is selected from the group consisting of mutation R178W, L713 splice, Q1272K, L479I, L1295F, E1024K, P1467S, G737R, K1023T, G966C, S1512fs in COL14A1; the mutation in TNN is selected from the group consisting of mutation V353M, Y296S, A733P, D707Y, D471Y, P1010T, S71L, D457Y, P1155L, R476C, Q872H, Q261L, D798Y, C1237*, D67N and T823S in TNN; the mutation in TP53 is selected from the group consisting of mutation Q331R, C135fs, E285K, V274F, Y220C, P250L, R175H, R248Q, R280K, R248L, C176Y, A307_splice, R273L, R158L, A138fs, H193R, A159D, C277F, R248W, Y220C, V274F, R196*, E224_splice, K164*, M246I, A159V, S241F, C242R, S261_splice, E339* in TP53.

In certain embodiments, the method further comprises recommending the patient who is identified as a likely non-responder not to be treated with a monotherapy of the ERK inhibitor, or not to be treated with an MEK inhibitor.

In certain embodiments, the method further comprises recommending the patient who is identified as a likely non-responder to be treated with a different MEK inhibitor, or to be treated with a combined therapy of a different MEK inhibitor and an additional therapeutic agent of distinct mechanism.

In certain embodiments, the method further comprises recommending the patient who is identified as a likely responder to be treated with the MEK inhibitor.

In certain embodiments, the sample is a cancer cell or tissue derived from the patient.

In another aspect, the present disclosure provides a method for predicting the responsiveness of a cancer cell to a ERK inhibitor, comprising detecting the presence of at least one mutation in one or more genes selected from the group consisting of ADAM12, PEX5L, TNN and TP53, in the cancer cell, by contacting a nucleic acid sample derived from the cancer cell with at least one oligonucleotide which allows specific detection of the mutation; wherein presence of the mutation in ADAM12, PEX5L, TNN, TP53 and/or any combination thereof is indicative of decreased responsiveness of the cancer cell to the ERK inhibitor.

In certain embodiments, the cancer cell is derived from a cancer patient.

In certain embodiments, the ERK inhibitor is SCH772984.

In certain embodiments, the mutation in ADAM12 is selected from the group consisting of mutation Q650K, R240L, C440Y, Q228E, H247D, M322I, T97fs, P168L and G308E in ADAM12; the mutation in PEX5L is selected from the group consisting of mutation D179N, S229Y, G4E, T89K, Q355E, D39N, L571F, D113N in PEX5L; the mutation in TNN is selected from the group consisting of mutation V353M, Y296S, A733P, D707Y, D471Y, P1010T, S71L, D457Y, P1155L, R476C, Q872H, Q261L, D798Y, C1237*, D67N and T823S in TNN; the mutation in TP53 is selected from the group consisting of mutation Q331R, C135fs, E285K, V274F, Y220C, P250L, R175H, R248Q, R280K, R248L, C176Y, A307_splice, R273L, R158L, A138fs, H193R, A159D, C277F, R248W, Y220C, V274F, R196*, E224_splice, K164*, M246I, A159V, S241F, C242R, S261_splice, E339* in TP53.

In certain embodiments, the detecting step comprises amplifying at least a portion of the gene with the oligonucleotide as primer, and detecting the amplification product and thereby determining the presence of the mutation in the gene.

In certain embodiments, the detecting step comprises contacting the nucleic acid sample with the oligonucleotide which specifically hybridizes to the mutation of the gene to form a complex, and detecting the formation of the complex and thereby determining the presence of the mutation in the gene.

In yet another aspect, the present disclosure provides a method of identifying a likely responder or a likely non-responder to an ERK inhibitor, comprising detecting the presence of at least one mutation in one or more genes selected from the group consisting of ADAM12, PEX5L, TNN and TP53, in a sample from the patient, by contacting the sample with at least one oligonucleotide which allows specific detection of the mutation; identifying the patient as a likely non-responder to the ERK inhibitor if at least one mutation in ADAM12, PEX5L, TNN, TP53 and/or any combination thereof is detected in the sample.

In certain embodiments, the ERK inhibitor is SCH772984.

In certain embodiments, the mutation in ADAM12 is selected from the group consisting of mutation Q650K, R240L, C440Y, Q228E, H247D, M322I, T97fs, P168L and G308E in ADAM12; the mutation in PEX5L is selected from the group consisting of mutation D179N, S229Y, G4E, T89K, Q355E, D39N, L571F, D113N in PEX5L; the mutation in TNN is selected from the group consisting of mutation V353M, Y296S, A733P, D707Y, D471Y, P1010T, S71L, D457Y, P1155L, R476C, Q872H, Q261L, D798Y, C1237*, D67N and T823S in TNN; the mutation in TP53 is selected from the group consisting of mutation Q331R, C135fs, E285K, V274F, Y220C, P250L, R175H, R248Q, R280K, R248L, C176Y, A307_splice, R273L, R158L, A138fs, H193R, A159D, C277F, R248W, Y220C, V274F, R196*, E224_splice, K164*, M246I, A159V, S241F, C242R, S261_splice, E339* in TP53.

In certain embodiments, the method further comprises recommending the patient who is identified as a likely non-responder not to be treated with a monotherapy of the ERK inhibitor, or not to be treated with an ERK inhibitor.

In certain embodiments, the method further comprises recommending the patient who is identified as a likely non-responder to be treated with a different ERK inhibitor, or to be treated with a combined therapy of a different ERK inhibitor and an additional therapeutic agent of distinct mechanism.

In certain embodiments, the sample is a cancer cell or tissue derived from the patient.

In another aspect, the present disclosure provides a kit comprising at least one oligonucleotide useful for determining the presence of at least one mutation in one or more genes selected from the group consisting of ADAM12, COL14A1, TNN, TP53, ITGB, and PEX5L.

In certain embodiments, the at least one oligonucleotide comprises a first oligonucleotide useful for determining the presence of at least one mutation in ADAM12, a second oligonucleotide useful for determining the presence of at least one mutation in COL14L1, a third oligonucleotide useful for determining the presence of at least one mutation in TNN, a fourth oligonucleotide useful for determining the presence of at least one mutation in TP53, or any combination thereof.

In certain embodiments, the at least one oligonucleotide comprises a first oligonucleotide useful for determining the presence of at least one mutation in ADAM12, a second oligonucleotide useful for determining the presence of at least one mutation in PEX5L, a third oligonucleotide useful for determining the presence of at least one mutation in TNN, a fourth oligonucleotide useful for determining the presence of at least one mutation in TP53, and or combination thereof.

In certain embodiments, the at least one oligonucleotide comprises a pair of primer useful for amplifying at least a portion of the gene sequence, or comprises a probe useful for specifically hybridizing to the mutation of the gene to form a complex.

In another aspect, the present disclosure provides use of at least one oligonucleotide in the manufacture of a kit for predicting the responsiveness of a cancer cell or a cancer patient to an MEK inhibitor or a ERK inhibitor, wherein the oligonucleotide is useful for detecting the presence of at least one mutation in one or more genes selected from the group consisting of ADAM12, COL14A1, TNN, TP53, and PEX5L.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the increased sensitivity to MEK inhibitor Trametinib in cells harboring mutations in ADAM12 gene.

FIG. 2 illustrates the decreased sensitivity to MEK inhibitor Trametinib in cells harboring mutations in COL14A1 gene.

FIG. 3 illustrates the increased sensitivity to MEK inhibitor Trametinib in cells harboring mutations in TNN gene.

FIG. 4 illustrates the increased sensitivity to MEK inhibitor Trametinib in cells harboring mutations in TP53 gene.

FIG. 5 illustrates the increased sensitivity to MEK inhibitor Trametinib in cells harboring multiple mutations in ADAM12, COL14A1, TNN, and TP53 gene.

FIG. 6 illustrates the increased sensitivity to ERK inhibitor SCH772984 in cells harboring mutations in ADAM12 gene.

FIG. 7 illustrates the increased sensitivity to ERK inhibitor SCH772984 in cells harboring mutations in PEX5L gene.

FIG. 8 illustrates the increased sensitivity to ERK inhibitor SCH772984 in cells harboring mutations in TNN gene.

FIG. 9 illustrates the increased sensitivity to ERK inhibitor SCH772984 in cells harboring mutations in TP53 gene.

FIG. 10 illustrates the increased sensitivity to ERK inhibitor SCH772984 in cells harboring multiple mutations in ADAM 12, PEX5L, TNN and TP53 gene.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the present disclosure provides a method for predicting the responsiveness of a cancer cell to an MEK inhibitor. In certain embodiments, the method comprises detecting the presence of at least one mutation in one or more genes selected from the group consisting of ADAM12, COL14A1, TNN, and TP53, in the cancer cell, by contacting a nucleic acid sample derived from the cancer cell with at least one oligonucleotide which allows specific detection of the mutation; wherein presence of mutation in ADAM12, COL14A1, TNN, TP53 and/or any combination thereof is indicative of decreased responsiveness of the cancer cell to the ERK inhibitor.

The mitogen-activated extracellular signal-regulated kinase (MEK)-extracellular regulated protein kinases (ERK) cascade, also known as Ras-Raf-MEK-ERK signaling pathway, is one of the key signaling pathways involved in tumor oncogenic growth and progression. The signal pathway starts when a signaling molecule (e.g., a growth factor) binds to the receptor on the cell surface. This triggers Ras (a GTPase) to sap its GDP for a GTP. The GTP-bound Ras then activate Raf, which activates MEK, which activates ERK. ERK then activates some proteins, such as myc, that control cell division and cell survival. When one or more proteins in the pathway, such as Ras, are mutated, it can lead to the signaling pathway stuck in the activated status, which is a necessary step in the development of many cancers. As a result, inhibitors of the MEK/ERK signaling pathway have been developed to treat cancer. Certain patients have been found resistant to MEK or ERK inhibitors. The mechanisms underlying resistance to these inhibitors are unclear.

Multiple MEK and ERK1/2 inhibitors are currently under clinical investigation for cancer treatment and more agents targeting MEK or ERK1/2 are under preclinical development. Examples of MEK inhibitors include without limitation Trametinib (GSK1120212), Selumetinib, Binimetinib (MEK162), PD-325901, Cobimetinib (GDC-0973, XL518), and CI-1040 (PD035901). ERK inhibitors include without limitation SCH772984, FR180204, GDC-0994.

In certain embodiments, the MEK inhibitor is Trametinib. Trametinib (trade name Mekinist) has chemical name N-(3-{3-Cyclopropyl-5-[(2-fluoro-4-iodophenyl)amino]-6,8-dimethyl-2,4,7-trioxo-3,4,6,7-tetrahydropyrido[4,3-d]pyrimidin-1(2H)-yl}phenyl)acetamide. The structure of Trametinib is illustrated below.

As used herein, the term “responsiveness” refers to the likeliness of a cell/individual/patient/subject responding to the treatment of MEK or ERK inhibitor, i.e. showing decreased proliferation/growth and/or increased cell death after being treated with a MEK or ERK inhibitor. In certain embodiments, the responsiveness can be scaled as insensitive (i.e., less likely to respond), sensitive (likely to respond) and uncertain. In certain embodiments, the cell/individual/patient/subject is less likely to respond to a treatment of MEK or ER inhibitor when the cell/individual/patient/subject shows a decreased likeliness that a pathological complete response (pcR), i.e. absence of invasive cancer, will occur. In certain embodiments, the deceased likeliness means about 70%, 60%, 50%, 40%, 30%, 20%, 10% likeliness of the pcR occurred in a reference patient (e.g., a patient without mutation in the gene of interest). In certain embodiments, responsiveness of a cell can be evaluated by measuring IC50 of the cell to a MEK or ERK inhibitor.

As used herein, the term “mutation” refers to the deviation of a genomic DNA from a normal reference (e.g., wild-type genomic DNA), for example, additions, deletions, insertions, rearrangements, inversions, transitions, transversions, frame-shift mutations, nonsense mutations, missense mutations, translocations, and single nucleotide polymorphisms.

Methods of detecting the presence of a mutation in a gene are described herein and known in the art (in general, see e.g., Molecular Cloning A Laboratory Manual, 2nd Ed., ed. By Sambrook, Fritsch and Maniatis, Cold Spring Harbor Laboratory Press, 1989). Examples of the method include, without limitation, sequencing of nucleic acids (e.g., Sanger di-deoxy sequencing, “next generation” sequencing methods and single molecule sequencing), PCR (polymerase chain reaction)-based assay (real-time RCR, PCR-RFLP assay (see Cancer Research 59 (1999), 5169-5175), mass-spectrometric genotyping (e.g. MALDI-TOF), HPLC, enzymatic methods and SSPC (single strand conformation polymorphism analysis (see Pathol Int (1996) 46, 801-804)), hybridization-based assay (e.g., Northern-blot, Southern blot, 5′-exonuclease (TaqMan™) probe, molecular beacons, fluorescence energy transfer probes, Scorpion probes).

In certain embodiments, the method may include enzymatic amplification of DNA or cDNA fragments of the gene to be evaluated by PCR. The resulting PCR products may be subjected to either conventional Sanger-based dideoxy nucleotide sequencing methods or parallel sequencing methods (“next generation sequencing”) such as those marketed by Roche (454 technology), Illumina (Solexa technology) ABI (Solid technology) or Invitrogen (IonTorrent). Mutations may be identified from sequence reads by comparison with publicly available gene sequence databases. Alternatively, mutations may be identified by incorporation of allele-specific probes that can either be detected using enzymatic detection reactions, fluorescence, mass spectrometry or others.

In certain embodiments, the method may include amplifying DNA or cDNA fragments of the gene of interest using primers specifically bind to only one of normal and mutated sequence. As a result, amplification product can only be found in one of normal and mutated genes. As such, the presence of the mutation in the gene can be determined by detecting the presence of the amplification product.

In certain embodiments, the method may include contacting the nucleic acid sample with an oligonucleotide probe which specifically hybridizes to the mutation of the gene to form a complex. The oligonucleotide probe can be designed as not hybridizing to the normal sequence of the gene. The presence of the hybridization complex can be detected using reporter signals, e.g., fluorescence. As a result, the presence of the mutation in the gene can be determined by detecting the formation of the complex.

In certain embodiments, the mutation is present in an exon of the gene. In certain embodiment, the presence of the mutation leads to the amino acid change of the polypeptide encoded by the gene. Table 3 shows exemplary nucleic acid sequences of the mutations to be determined in accordance to the present invention. As used herein, a specific mutation is annotated as the resulted amino acid change. For example, mutation Q650K refers to a codon/triplet encoding amino acid K at position 650 of the gene, where amino acid G exists in wild type sequence.

ADAM12 gene (Gene ID: 8038) encodes a member of the ADAM (a disintegrin and metaloprotease) protein family. Members of the ADAM family are membrane-anchored proteins structurally related to snake venom disintegrins, and have been found involved in cell-cell and cell-matrix interactions. ADAM12 gene has two alternative spliced transcripts: a shorter secreted form and a longer member-bound form.

COL14A1 gene (Gene ID: 7373) encodes the alpha chain of type XIV collage, a member of the FACIT (fibril-associated collagens with interrupted triple helices) collagen family. Type XIV collagen interacts with the fibril surface and is involved in the regulation of fribrillogenesis.

TNN gene (Gene ID: 63923) encodes tenascin N precursor. Tenascin is a family of extracellular matrix glycoproteins, whose member has been found to in healing wounds and in the stroma of some tumors.

TP53 gene (Gene ID: 7157) encodes tumor protein p53, which is a tumor suppressor protein containing transcriptional activation, DNA binding, and oligomerization domains. Tumor protein p53 responds to diverse cellular stresses to regulate expression of target genes, thereby inducing cell cycle arrest, apoptosis, senescence, DNA repair, or changes in metabolism. Mutations in TP 53 gene have been found to associate with a variety of cancers. Alternative splicing of TP53 gene and the use of alternate promoters result in multiple transcript variants and isoforms. Additional isoforms have also been shown to result from the use of alternate translation initiation codons. As used herein, position number refers to the sequence of tumor protein p53 isoform a.

In certain embodiments, the mutation in ADAM12 is selected from the group consisting of mutation Q650K, R240L, C440Y, Q228E, H247D, M322I, T97fs, P168L and G308E in ADAM12; the mutation in COL14A1 is selected from the group consisting of mutation R178W, L713 splice, Q1272K, L479I, L1295F, E1024K, P1467S, G737R, K1023T, G966C, S1512fs in COL14A1; the mutation in TNN is selected from the group consisting of mutation V353M, Y296S, A733P, D707Y, D471Y, P1010T, S71L, D457Y, P1155L, R476C, Q872H, Q261L, D798Y, C1237*, D67N and T823S in TNN; the mutation in TP53 is selected from the group consisting of mutation Q331R, C135fs, E285K, V274F, Y220C, P250L, R175H, R248Q, R280K, R248L, C176Y, A307_splice, R273L, R158L, A138fs, H193R, A159D, C277F, R248W, Y220C, V274F, R196*, E224_splice, K164*, M246I, A159V, S241F, C242R, S261_splice, E339* in TP53, wherein “*” means the mutation leads to a stop condon, “fs” means the mutations leads to frame shift, “splice” means the mutation leads to alternative splice of mRNA.

The cancer cell maybe, for example, derived from lung cancer, non small cell lung (NSCL) cancer, bronchioloalviolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, colon cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, mesothelioma, hepatocellular cancer, biliary cancer, chronic or acute leukemia, lymphocytic lymphomas, neoplasms of the central nervous system (CNS), spinal axis tumors, brain stem glioma, glioblastoma multiforme, astrocytomas, schwanomas, ependymonas, medulloblastomas, meningiomas, squamous cell carcinomas, pituitary adenoma, including refractory versions of any of the above cancers, or a combination of one or more of the above cancers. In certain embodiments, the cancer cell is derived from a cancer patient.

In another aspect, the present disclosure provides a method of identifying a likely responder or a likely non-responder to an MEK inhibitor, comprising detecting the presence of at least one mutation in one or more genes selected from the group consisting of ADAM12, COL14A1, TNN, and TP53, in a sample from the patient, by contacting the sample with at least one oligonucleotide which allows specific detection of the mutation; identifying the patient as a likely non-responder to the MEK inhibitor if at least one mutation in ADAM12, COL14A1, TNN, TP53 and/or any combination thereof is detected in the sample.

As used herein, the term “responder” can refer to an individual/patient/subject that is more likely to respond to a treatment using a MEK or ERK inhibitor. “More likely to respond” as used herein refers to an increased likeliness that a pathological complete response will occur in a patient treated with a MEK or ERK inhibitor. The term “non-responder” can refer to an individual/patient/subject that is less likely to respond to a treatment using a MEK or ERK inhibitor. “Less likely to respond” as used herein refers to an decreased likeliness that a pathological complete response will occur in a patient treated with a MEK or ERK inhibitor.

In certain embodiments, in cases where it is assessed that the patient is a likely “responder,” said patient is recommended to be treated with an MEK or ERK inhibitor.

In cases where the patient is identified as a likely non-responder, said patient is recommended not to be treated with a monotherapy of the MEK or ERK inhibitor, or not to be treated with an MEK or ERK inhibitor.

In certain embodiments, wherein the patient is identified as a likely non-responder to a MEK inhibitor, the patient is recommended to be treated with a different MEK inhibitor, or to be treated with a combined therapy of a different MEK inhibitor and an additional therapeutic agent of distinct mechanism. Examples of an MEK inhibitor different from Trametinib include, without limitation, Selumetinib, Binimetinib (MEK162), PD-325901, Cobimetinib (GDC-0973, XL518), and CI-1040 (PD035901).

In certain embodiments, the additional therapeutic agent of distinct mechanism can be an agent targeting PI3K-Akt-mTOR signaling pathway. The agents targeting PI3K-Akt-mTOR signaling pathway are known in the art and comprise, without limitation, fused pyrimidine derivatives as disclosed in U.S. Pat. No. 8,022,205 B2 or fused pyrrolopyrimidine derivatives as disclosed in WO2009/099163.

In certain embodiments, the additional agent of distinct mechanism can include c-Met inhibitors (e.g., ARQ197 (taventinib, developed by Daichi Sankyo and ArQule), AMG458 (developed by Amgen), GSK1363089 (also known as XL880 or foretinib, developed GSK), crizotinib (also known as PF2341066, developed by Pfizer), PF04217903 (developed by Pfizer), INCB28060 (developed by Incyte), E7050 (developed by Eisai), MK-2461 (developed by Merck), BMS-777607 (developed by BMS), JNJ-38877605 (developed by Johnson & Johnson), XL184 (developed by BMS/Exelixis)).

In certain embodiments, the additional therapeutic agent of distinct mechanism can be chemotherapeutic agents (e.g., cyclophosphamide (CTX; e.g. Cytoxan®), chlorambucil (CHL; e.g. Leukeran®), cisplatin (CisP; e.g. Platinol®) busulfan (e.g. Myleran®), melphalan, carmustine (BCNU), streptozotocin, triethylenemelamine (TEM), mitomycin C)

In certain embodiments, the additional therapeutic agent of distinct mechanism can be anti-metabolites, such as methotrexate (MTX), etoposide (VP16; e.g. Vepesid®), 6-mercaptopurine (6MP), 6-thiocguanine (6TG), cytarabine (Ara-C), 5-fluorouracil (5-FU), capecitabine (e.g. Xeloda®), dacarbazine (DTIC)).

In certain embodiments, the additional therapeutic agent of distinct mechanism can be other antitumor agents, such as paclitaxel (e.g. Taxol®) and pactitaxel derivatives, the cytostatic agents, glucocorticoids such as dexamethasone (DEX; e.g. Decadron®) and corticosteroids such as prednisone, nucleoside enzyme inhibitors such as hydroxyurea, amino acid depleting enzymes such as asparaginase, leucovorin, folinic acid, raltitrexed, and other folic acid derivatives, and similar, diverse antitumor agents.

In certain embodiments, the additional therapeutic agent of distinct mechanism can be anti-hormonal agents (e.g., steroid receptor antagonists, anti-estrogens such as tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, other aromatase inhibitors, 42-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, and toremifene (e.g. Fareston®); anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above; agonists and/or antagonists of glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH) and LHRH (leuteinizing hormone-releasing hormone); the LHRH agonist goserelin acetate, commercially available as Zoladex® (AstraZeneca); the LHRH antagonist D-alaninamide N-acetyl-3-(2-naphthalenyl)-D-alanyl-4-chloro-D-phenylalanyl-3-(3-pyridinyl)-D-alanyl-L-seryl-N6-(3-pyridinylcarbonyl)-L-lysyl-N6-(3-pyridinylcarbonyl)-D-lysyl-L-leucyl-N6-(1-methylethyl)-L-lysyl-L-proline (e.g Antide®, Ares-Serono); the LHRH antagonist ganirelix acetate; the steroidal anti-androgens cyproterone acetate (CPA) and megestrol acetate, commercially available as Megace® (Bristol-Myers Oncology); the nonsteroidal anti-androgen flutamide (2-methyl-N-[4, 20-nitro-3-(trifluoromethyl)phenylpropanamide), commercially available as Eulexin® (Schering Corp.); the non-steroidal anti-androgen nilutamide, (5,5-dimethyl-3-[4-nitro-3-(trifluoromethyl-4′-nitrophenyl)-4,4-dimethyl-imidazolidine-dione); and antagonists for other non-permissive receptors, such as antagonists for RAR, RXR, TR, VDR, and the like).

In certain embodiments, the additional therapeutic agent of distinct mechanism can be angiogenesis inhibitors (e.g., VEGFR inhibitors, such as SU-5416 and SU-6668 (Sugen Inc. of South San Francisco, Calif., USA), or as described in, for example International Application Nos. WO 99/24440, WO 99/62890, WO 95/21613, WO 99/61422, WO 98/50356, WO 99/10349, WO 97/32856, WO 97/22596, WO 98/54093, WO 98/02438, WO 99/16755, and WO 98/02437, and U.S. Pat. Nos. 5,883,113, 5,886,020, 5,792,783, 5,834,504 and 6,235,764; VEGF inhibitors such as IM862 (Cytran Inc. of Kirkland, Wash., USA); angiozyme, a synthetic ribozyme from Ribozyme (Boulder, Colo.) and Chiron (Emeryville, Calif.); and antibodies to VEGF, such as bevacizumab (e.g. Avastin™ Genentech, South San Francisco, Calif.), a recombinant humanized antibody to VEGF; integrin receptor antagonists and integrin antagonists, such as to α_(v)β₃, α_(v)β₃₅ and α_(v)β₆ integrins, and subtypes thereof, e.g. cilengitide (EMD 121974), or the anti-integrin antibodies, such as for example α_(v)β₃₃ specific humanized antibodies (e.g. Vitaxin®); factors such as IFN-alpha (U.S. Pat. Nos. 41,530,901, 4,503,035, and 5,231,176); angiostatin and plasminogen fragments (e.g. kringle 14, kringle 5, kringle 1-3 (O'Reilly, M. S. et al. (1994) Cell 79:315-328; Cao et al. (1996) J. Biol. Chem. 271: 29461-29467; Cao et al. (1997) J. Biol. Chem. 272:22924-22928); endostatin (O'Reilly, M. S. et al. (1997) Cell 88:277; and International Patent Publication No. WO 97/15666); thrombospondin (TSP-1; Frazier, (1991) Curr. Opin. Cell Biol. 3:792); platelet factor 4 (PF4); plasminogen activator/urokinase inhibitors; urokinase receptor antagonists; heparinases; fumagillin analogs such as TNP-4701; suramin and suramin analogs; angiostatic steroids; bFGF antagonists; flk-1 and flt-1 antagonists; anti-angiogenesis agents such as MMP-2 (matrix-metalloprotienase 2) inhibitors and MMP-9 (matrix-metalloprotienase 9) inhibitors).

In certain embodiments, the sample is a cancer cell or tissue derived from the patient.

In another aspect, the present disclosure provides a method for predicting the responsiveness of a cancer cell to a ERK inhibitor, comprising detecting the presence of at least one mutation in one or more genes selected from the group consisting of ADAM12, PEX5L, TNN and TP53, in the cancer cell, by contacting a nucleic acid sample derived from the cancer cell with at least one oligonucleotide which allows specific detection of the mutation; wherein presence of the mutation in ADAM12, PEX5L, TNN, TP53 and/or any combination thereof is indicative of decreased responsiveness of the cancer cell to the ERK inhibitor.

In certain embodiments, the cancer cell is derived from a cancer patient.

In certain embodiments, the ERK inhibitor is SCH772984. SCH772984, with chemical name (R)-1-(2-oxo-2-(4-(4-(pyrimidin-2-yl)phenyl)piperazin-1-yl)ethyl)-N-(3-(pyridin-4-yl)-1H-indazol-5-yl)pyrrolidine-3-carboxamide, is a novel, selective and ATP competitive inhibitor of ERK1/2 (see Morris E J et al., Discovery of a novel ERK inhibitor with activity in models of acquired resistance to BRAF and MEK inhibitors, Cancer Discov. 20133(7): 742-50). The structure of SCH772984 is illustrated as below.

In certain embodiments, the mutation in ADAM12 is selected from the group consisting of mutation Q650K, R240L, C440Y, Q228E, H247D, M322I, T97fs, P168L and G308E in ADAM12; the mutation in PEX5L is selected from the group consisting of mutation D179N, S229Y, G4E, T89K, Q355E, D39N, L571F, D113N in PEX5L; the mutation in TNN is selected from the group consisting of mutation V353M, Y296S, A733P, D707Y, D471Y, P1010T, S71L, D457Y, P1155L, R476C, Q872H, Q261L, D798Y, C1237*, D67N and T823S in TNN; the mutation in TP53 is selected from the group consisting of mutation Q331R, C135fs, E285K, V274F, Y220C, P250L, R175H, R248Q, R280K, R248L, C176Y, A307_splice, R273L, R158L, A138fs, H193R, A159D, C277F, R248W, Y220C, V274F, R196*, E224_splice, K164*, M246I, A159V, S241F, C242R, S261_splice, E339* in TP53.

In certain embodiments, the detecting step comprises amplifying at least a portion of the gene with the oligonucleotide as primer, and detecting the amplification product and thereby determining the presence of the mutation in the gene.

In certain embodiments, the detecting step comprises contacting the nucleic acid sample with the oligonucleotide which specifically hybridizes to the mutation of the gene to form a complex, and detecting the formation of the complex and thereby determining the presence of the mutation in the gene.

In yet another aspect, the present disclosure provides a method of identifying a likely responder or a likely non-responder to an ERK inhibitor, comprising detecting the presence of at least one mutation in one or more genes selected from the group consisting of ADAM12, PEX5L, TNN and TP53, in a sample from the patient, by contacting the sample with at least one oligonucleotide which allows specific detection of the mutation; identifying the patient as a likely non-responder to the ERK inhibitor if at least one mutation in ADAM12, PEX5L, TNN, TP53 and/or any combination thereof is detected in the sample.

In certain embodiments, the method further comprises recommending the patient who is identified as a likely non-responder not to be treated with a monotherapy of the ERK inhibitor, or not to be treated with an ERK inhibitor.

In certain embodiments, the method further comprises recommending the patient who is identified as a likely non-responder to be treated with a different ERK inhibitor, or to be treated with a combined therapy of a different ERK inhibitor and an additional therapeutic agent of distinct mechanism. Examples of ERK inhibitors other than SCH772984 include FR180204, GDC-0994.

In another aspect, the present disclosure provides a kit comprising at least one oligonucleotide useful for determining the presence of at least one mutation in one or more genes selected from the group consisting of ADAM12, COL14A1, TNN, TP53, ITGB, and PEX5L.

In certain embodiments, the at least one oligonucleotide comprises a first oligonucleotide useful for determining the presence of at least one mutation in ADAM12, a second oligonucleotide useful for determining the presence of at least one mutation in COL14L1, a third oligonucleotide useful for determining the presence of at least one mutation in TNN, a fourth oligonucleotide useful for determining the presence of at least one mutation in TP53, or any combination thereof.

In certain embodiments, the at least one oligonucleotide comprises a first oligonucleotide useful for determining the presence of at least one mutation in ADAM12, a second oligonucleotide useful for determining the presence of at least one mutation in PEX5L, a third oligonucleotide useful for determining the presence of at least one mutation in TNN, a fourth oligonucleotide useful for determining the presence of at least one mutation in TP53, and or combination thereof.

In certain embodiments, the at least one oligonucleotide comprises a pair of primer useful for amplifying at least a portion of the gene sequence, or comprises a probe useful for specifically hybridizing to the mutation of the gene to form a complex.

In another aspect, the present disclosure provides use of at least one oligonucleotide in the manufacture of a kit for predicting the responsiveness of a cancer cell or a cancer patient to an ERK inhibitor or a MEK inhibitor, wherein the oligonucleotide is useful for detecting the presence of at least one mutation in one or more genes selected from the group consisting of ADAM12, COL14A1, TNN, TP53, and PEX5L.

Example 1

The following is an example of identifying genes correlated with sensitivity to MEK inhibitors and/or ERK inhibitors.

We examined the anti-proliferation activity of a MEK inhibitor, trametinib, and an ERK1/2 inhibitor, SCH772984, in a panel of 50 cell lines (see Table 1).

TABLE 1 Cell lines used in the screen Cancer Ras/raf Type No. Cell line mutation Growth P. Medium 1. 1.1 BT474 150 Adherent DMEM + 0.01 mg/ml bovine insulin Breast 1.2 DU4475 423 Suspension RPMI-1640 1.3 MDA-MB-231 576 Adherent L15 1.4 ZR-75-1 493 Adherent RPMI-1640 2. 2.1 COLO 205 591 Adherent RPMI-1640 Colorectal 2.2 DLD-1 164 Adherent RPMI-1640 2.3 HCT-116 444 Adherent McCoys'5a 2.4 HCT-15 627 Adherent RPMI-1640 2.5 HCT-8 354 Adherent RPMI-1640 2.6 HT-29 504 Adherent McCoys'5a 2.7 KM12 L4 779 Adherent DMEM 2.8 LoVo 737 Adherent F12K 2.9 LS513 775 Adherent RPMI-1640 2.1 RKO 44 Adherent MEM 2.11 SW1116 42 Adherent L15 2.12 SW480 237 Adherent L15 2.13 SW620 590 Adherent L15 3. 3.1 Hep G2 243 Adherent EMEM Liver 3.2 HuCCT1 396 Adherent RPMI-1640 3.3 SK-HEP-1 171 Adherent MEM + 0.1 mMNEAA 3.4 SNU-387 287 Adherent RPMI-1640 4. 4.1 A549 101 Adherent F12K Lung 4.2 Calu-6 254 Adherent EMEM 4.3 NCI-H1155 584 Suspension ACL-4 4.4 NCI-H1299 577 Adherent RPMI-1640 4.5 NCI-H1373 360 Adherent RPMI-1640 4.6 NCI-H1395 386 Adherent RPMI-1640 4.7 NCI-H1573 426 Adherent ACL-4 4.8 NCI-H1651 429 Adherent ACL-4 4.9 NCI-H1666 425 Adherent ACL-4 4.1 NCI-H1792 400 Adherent RPMI-1640 4.11 NCI-H2009 513 Adherent HITES + 10% FBS 4.12 NCI-H2227 501 Adherent HITES + 10% FBS 4.13 NCI-H23 380 Adherent RPMI-1640 4.14 NCI-H358 571 Adherent RPMI-1640 4.15 NCI-H441 759 Adherent RPMI-1640 4.16 NCI-H460 24 Adherent RPMI-1640 4.17 SK-LU-1 403 Adherent EMEM 4.18 SW1271 471 Adherent L15 5. 5.1 AsPC-1 736 Adherent RPMI-1640 Pancreas 5.2 Capan-1 731 Adherent IMDM + 20% FBS 5.3 CFPAC-1 43 Adherent IMDM + 20% FBS 5.4 MIA PaCa-2 167 Adherent DMEM + 10% FBS + 2.5% HS 5.5 PANC-1 156 Adherent DMEM 6. 6.1 PL45 422 Adherent DMEM Skin 6.2 A2058 420 Adherent DMEM 6.3 A-375 716 Adherent DMEM 6.4 SK-MEL-5 154 Adherent MEM + 10% FBS + 0.01 mM NEAA 7. 7.1 AGS 295 Adherent F12K Stomach 7.2 SNU-1 292 Suspension RPMI-1640 7.3 SNU-719 552 Adherent RPMI-1640

Materials and Methods

Cell Culture

All the cells will be cultured in the media supplemented with 10% FBS except for which are marked specially, in the temperature of 37° C., 5% CO₂ and 95% humidity.

Cell Viability Reagent

Cell viability is assayed by using CellTiter-Glo® Luminescent Cell Viability Assay Kit (Cat. No.: G7572, Promega. Store at −20° C.). To prepare the CellTiter_Glo Reagent, the CellTiter-Glo Buffer was thawed and equilibrated to room temperature prior to use. For convenience the CellTiter-Glo Buffer may be thawed and stored at room temperature for up to 48 hours prior to use. The lyophilized CellTiter-Glo Substrate is equilibrated to room temperature prior to use. The appropriate volume (100 ml) of CellTiter-Glo Buffer is transferred into the amber bottle containing CellTiter-Glo Substrate to reconstitute the lyophilized enzyme/substrate mixture, which forms the CellTiter-Glo Reagent. In certain cases, the entire liquid volume of the CellTiter-Glo Buffer bottle may be added to the CellTiter-Glo Substrate vial. Mix by gently vortexing, swirling or by inverting the contents to obtain a homogeneous solution. The CellTiter-Glo Substrate should go into solution easily in less than one minute.

MEK and ERK Inhibitor

MEK inhibitor Trametinib was purchased from Selleckchem (Cat No. 52673) and stored at −20° C. before use. ERK1/2 inhibitor SCH772984 was purchased from Selleckchem (Cat No. 57101) and stored at −20° C. before use.

Equipment

The following equipment was used in the experiments: EnVision Multi Label Reader 2104-0010A, PerkinElmer (USA); Countstar, Inno-Alliance Biotech (USA); Forma Series II Water Jacket CO2 Incubator, Thermo Scientific (USA); Biological safety Cabinet, Thermo Scientific, (USA); Inverted Microscope, Olympus CKX41SF (Japan).

Cytotoxicity and IC50 Determination

The day before the experiment (Day −1), cells were dissociated during the logarithmic growth period with Cell Disassociation Buffer (Gibco 13151-014) and mixed with appropriate cell media and centrifuge at 1000 rpm for 3 minutes. The cells were re-suspended and counted using Countstar before adjusting cell concentrations to optimized density (i.e. 4.44×10⁴ cells/ml) with respective culture medium listed in Table 1 for 3-day CTG assay (The cell density was optimized before actual study; cell density used in the test may vary for different cell lines). 90 μl cell suspensions were added to two 96-well plates (plates A and B) with the final cell density of 4×10³ cells/well for 3-day CTG assay (the cell density was optimized before actual study; cell density used in the test may vary for different cell lines). The plate A and B group were incubated for overnight in humidified incubator at 37° C. with 5% CO₂.

On Day 0, for plate A group, 10 μl culture medium was added to each well for T0 reading. CellTiter-Glo® Reagent was added at equal volume of cell culture medium present in each well (e.g., add 100 μl of reagent to 100 μl of medium containing cells for a 96-well plate). Contents were mixed for 2 minutes on an orbital shaker to facilitate cell lysis. The plate was allowed to incubate at room temperature for 10 minutes to stabilize luminescent signal. Backseal black sticker was added to the bottom of each plate. Luminescence was recorded using EnVision Multi Label Reader. This formed the basis for T0 value.

On Day 0, the test articles and positive controls were dissolved at the concentration indicated at Test Article Dilution map. 100× solution in PBS was prepared and then diluted with appropriate culture media (1:10) into 10×working solutions. 10 μl (10×) drug solutions were dispensed in each well (triplicate for each drug concentration) of the plate B group according to plate inoculation map. The test plates were incubated for 4 days in the humidified incubator at 37° with 5% CO₂.

On Day 3, CellTiter-Glo® Reagent was added at equal volume of cell culture medium present in each well (e.g., add 100 μl of reagent to 100 μl of medium containing cells for a 96-well plate). Contents were mixed for 2 minutes on an orbital shaker to induce cell lysis. The plate was allowed to incubate at room temperature for 10 minutes to stabilize luminescent signal. Backseal black sticker was placed to the bottom of each plate. Luminescence was recorded using EnVision Multi Label Reader.

The data were displayed graphically using GraphPad Prism 5.0. In order to calculate IC50s, a dose-response curve was fitted using a nonlinear regression model with a sigmoidal dose response. The formula for calculating surviving rate was shown below; Absolute IC50 is calculated where Y axis set at 50% using GraphPad Prism 5.0. Software.

The surviving rate (%)=(Lum_(Test article)−Lum_(Medium control))/(Lum_(None treated)−Lum_(Medium control))×100%.

Lum_(None treated)−Lum_(Medium control) is set as 100% and Lum_(Medium control) is set for 0% surviving rate. T0 value was presented as percentage of Lum_(None treated).

Statistical Analysis

We divided the 63 cell lines into sensitive, insensitive, and uncertain groups according to their IC50's for SCH772984 and Trametinib, respectively, then detected genes with differential expression or different mutation types between sensitive and insensitive groups. These genes were enriched in several cancer related pathways.

The 63 cell lines were divided into 3 groups (See Table 1): a sensitive group (IC50 values were less than 1), an insensitive group (IC50 values are greater than 10), and an uncertain group (the rest). Accordingly, we got 25 sensitive and 22 insensitive cell lines for SCH772984, and 34 sensitive and 24 insensitive cell lines for Trametinib. Only cell lines with genomic data were used in subsequent analysis. The differentially expressed genes and enriched pathways were analysed using GSEA software, the genes with different mutation types were detected using Fisher's exact test.

Results

For Trametinib, 32 sensitive and 23 insensitive cell lines have gene expression profiled by Affymetrix U219 arrays, 34 gene sets are significantly enriched at nominal p-value <1% (See Table S2). 29 sensitive and 20 insensitive cell lines have mutation information, and ADAM12, COL14A1, TNN and TP53 were identified by P-value cutoff of 0.01 (See Table 3 and FIG. 1-5).

For SCH772984, 23 sensitive and 22 insensitive cell lines have gene expression profiled by Affymetrix U219 arrays, 10 gene sets are significantly enriched at nominal p-value <1%. 21 sensitive and 18 insensitive cell lines have mutation information, and ADAM12, PEX5L, TNN and TP53 were identified by P-value cutoff of 0.01 (See Table 3 and FIG. 6-10).

TABLE 2 IC50 information Absolute IC50 (uM) Number Cell line SCH772984 Trametinib 1 A549 1.78 0.24 2 A2058 NA 0.12 3 Calu6 0.56 0.14 4 DLD-1 52.77  6.52 5 HCT116 0.45 0.04 6 HepG2 0.13 0.00 7 MDA-MB-231 32.28  23.98  8 NCI-H23 3.69 0.30 9 NCI-H460 5.53 NA 10 RKO 17.46  6.70 11 SW620 0.26 0.01 12 SW480 NA 6.65 13 HCT-8 1.27 0.05 14 HCT-15 15.57  29.33  15 HT29 0.23 0.01 16 LoVo 0.41 0.11 17 LS513 0.78 0.01 18 NCI-H358 0.52 0.05 19 NCI-H441 NA 324.66  20 NCI-H1299 NA 0.91 21 NCI-H1792 4.70 0.16 22 PANC-1 64.31  330.05  23 Sk-Hep-1 5.29 29.14  24 Sk-Mel-5 0.20 0.01 25 BT474 NA 39.00  26 Colo205 0.04 0.00 27 KM12L4 0.36 0.01 28 MIAPaCa2 0.21 0.03 29 NCI-H1155 NA 636.34  30 NCI-H1373 NA NA 31 NCI-H1651 48.79  NA 32 NCI-H1666 0.92 0.13 33 NCI-H2009 4.36 0.41 34 NCI-H2227 28.74  NA 35 PL45 0.66 0.12 36 SK-LU-1 4.69 NA 37 SNU-1 0.64 0.10 38 ZR-75-1 NA NA 39 22RV1 5.01 202.02  40 BxPc-3 0.32 0.03 41 HCC2935 NA NA 42 HCC4006 2.77 0.41 43 Hela 5.38 NA 44 Hep3B 0.25 0.05 45 HM-7 0.79 0.39 46 HT1376 4.27 NA 47 KYSE150 1.72 5.28 48 NCI-H1703 NA 14.99  49 PC-3 14.29  NA 50 Du145 NA NA

TABLE 3 Gene Mutations Detected in MEK/ERK inhibitor insensitive cell lines. Variant Variant Tumor Gene Classification Type Sample Genome Change ADAM12 Missense SNP DU145 g.chr10: 127734680G > T ADAM12 Missense SNP DU145 g.chr10: 127797193C > A ADAM12 Missense SNP NCIH1573 g.chr10: 127760059C > T ADAM12 Missense SNP NCIH1573 g.chr10: 127797230G > C ADAM12 Missense SNP NCIH1703 g.chr10: 127797173G > C ADAM12 Missense SNP RKO g.chr10: 127787024C > T ADAM12 Frame Shift Del DEL RKO g.chr10: 127843846_127843846delT ADAM12 Missense SNP SW480 g.chr10: 127806716G > A ADAM12 Missense SNP HCT15 g.chr10: 127787067C > T COL14A1 Missense SNP 22RV1 g.chr8: 121209125C > T COL14A1 Splice Site SNP SNP DU145 g.chr8: 121239592G > T COL14A1 Missense SNP DU145 g.chr8: 121293288C > A COL14A1 Missense SNP NCIH1573 g.chr8: 121222108C > A COL14A1 Missense SNP NCIH2009 g.chr8: 121295933C > T COL14A1 Missense SNP NCIH441 g.chr8: 121279119G > A COL14A1 Missense SNP NCIH460 g.chr8: 121313055C > T COL14A1 Missense SNP RKO g.chr8: 121243717G > A COL14A1 Missense SNP SNU719 g.chr8: 121279117A > C COL14A1 Missense SNP HCT15 g.chr8: 121275133G > T COL14A1 Frame Shift Del DEL NCIH1373 g.chr8: 121326250_121326250delC TNN Missense SNP A549 g.chr1: 175052894G > A TNN Missense SNP DU145 g.chr1: 175049401A > C TNN Missense SNP NCIH1573 g.chr1: 175086152G > C TNN Missense SNP NCIH1703 g.chr1: 175067731G > T TNN Missense SNP NCIH2009 g.chr1: 175063212G > T TNN Missense SNP A2058 g.chr1: 175105993C > T TNN Missense SNP HCT15 g.chr1: 175063227C > T TNN Missense SNP HCT15 g.chr1: 175087926G > T TNN Missense SNP NCIH1299 g.chr1: 175063170G > T TNN Missense SNP NCIH1373 g.chr1: 175046753G > A TNN Missense SNP NCIH1373 g.chr1: 175087777A > T TNN Missense SNP NCIH2227 g.chr1: 175048841A > T TNN Missense SNP NCIH2227 g.chr1: 175087702G > T TNN Nonsense SNP NCIH2227 g.chr1: 175113638C > A TNN Missense SNP NCIH441 g.chr1: 175096204C > A TNN Missense SNP SKLU1 g.chr1: 175046766C > T TP53 Missense SNP 22RV1 g.chr17: 7576854T > C TP53 Frame Shift Del DEL ASPC1 g.chr17: 7578527_7578527delA TP53 Missense SNP BT474 g.chr17: 7577085C > T TP53 Missense SNP A2058 g.chr17: 7577118C > A TP53 Missense SNP BXPC3 g.chr17: 7578190T > C TP53 Nonsense SNP CALU6 g.chr17: 7578263G > A TP53 Missense SNP CAPAN1 g.chr17: 7578454G > A TP53 Missense SNP CFPAC1 g.chr17: 7577557A > G TP53 Missense SNP DU145 g.chr17: 7577118C > A TP53 Missense SNP HCC2935 g.chr17: 7578190T > C TP53 Missense SNP HT1376 g.chr17: 7577532G > A TP53 Missense SNP MDAMB231 g.chr17: 7577099C > T TP53 Missense SNP MIAPACA2 g.chr17: 7577539G > A TP53 Missense SNP NCIH1651 g.chr17: 7578403C > T TP53 Splice Site SNP SNP NCIH1703 g.chr17: 7577018C > A TP53 Splice Site SNP SNP NCIH1792 g.chr17: 7578176C > T TP53 Splice Site SNP SNP NCIH2227 g.chr17: 7577157T > G TP53 Missense SNP NCIH23 g.chr17: 7577543C > G TP53 Missense SNP NCIH441 g.chr17: 7578457C > A TP53 Frame Shift Del DEL PC3 g.chr17: 7578516_7578516delG TP53 Missense SNP SKLU1 g.chr17: 7578271T > C TP53 Nonsense SNP SNU387 g.chr17: 7578440T > A TP53 Missense SNP HUCCT1 g.chr17: 7578406C > T TP53 Missense SNP NCIH1573 g.chr17: 7577538C > A TP53 Missense SNP NCIH2009 g.chr17: 7577120C > A TP53 Missense SNP SW1116 g.chr17: 7578454G > T TP53 Missense SNP SW1271 g.chr17: 7577108C > A TP53 Missense SNP MIAPACA2 g.chr17: 7577539G > A PEX5L Missense SNP BT474 g.chr3: 179593236C > T PEX5L Missense SNP DU145 g.chr3: 179592155G > T PEX5L Missense SNP NCIH1573 g.chr3: 179754377C > T PEX5L Missense DNP NCIH2009 g.chr3: 179605504_179605505GG > TT PEX5L Missense SNP NCIH441 g.chr3: 179533669G > C PEX5L Missense SNP NCIH441 g.chr3: 179616013C > T PEX5L Missense SNP SW1271 g.chr3: 179519784C > A PEX5L Missense SNP NCIH1299 g.chr3: 179597885C > T Gene cDNA_Change Codon_Change Protein_Change ADAM12 c.1948C > A c.(1948-1950)CAA > AAA p.Q650K ADAM12 c.719G > T c.(718-720)CGA > CTA p.R240L ADAM12 c.1319G > A c.(1318-1320)TGT > TAT p.C440Y ADAM12 c.682C > G c.(682-684)CAG > GAG p.Q228E ADAM12 c.739C > G c.(739-741)CAC > GAC p.H247D ADAM12 c.966G > A c.(964-966)ATG > ATA p.M322I ADAM12 c.289_289delA c.(289-291)ACCfs p.T97fs ADAM12 c.503C > T c.(502-504)CCA > CTA p.P168L ADAM12 c.923G > A c.(922-924)GGG > GAG p.G308E COL14A1 c.532C > T c.(532-534)CGG > TGG p.R178W COL14A1 c.2137_splice p.L713_splice COL14A1 c.3814C > A c.(3814-3816)CAG > AAG p.Q1272K COL14A1 c.1435C > A c.(1435-1437)CTA > ATA p.L479I COL14A1 c.3883C > T c.(3883-3885)CTT > TTT p.L1295F COL14A1 c.3070G > A c.(3070-3072)GAA > AAA p.E1024K COL14A1 c.4399C > T c.(4399-4401)CCA > TCA p.P1467S COL14A1 c.2209G > A c.(2209-2211)GGA > AGA p.G737R COL14A1 c.3068A > C c.(3067-3069)AAA > ACA p.K1023T COL14A1 c.2896G > T c.(2896-2898)GGT > TGT p.G966C COL14A1 c.4535_4535delC c.(4534-4536)TCCfs p.S1512fs TNN c.1057G > A c.(1057-1059)GTG > ATG p.V353M TNN c.887A > C c.(886-888)TAC > TCC p.Y296S TNN c.2197G > C c.(2197-2199)GCC > CCC p.A733P TNN c.2119G > T c.(2119-2121)GAC > TAC p.D707Y TNN c.1411G > T c.(1411-1413)GAC > TAC p.D471Y TNN c.3464C > T c.(3463-3465)CCA > CTA p.P1155L TNN c.1426C > T c.(1426-1428)CGC > TGC p.R476C TNN c.2616G > T c.(2614-2616)CAG > CAT p.Q872H TNN c.1369G > T c.(1369-1371)GAC > TAC p.D457Y TNN c.199G > A c.(199-201)GAC > AAC p.D67N TNN c.2467A > T c.(2467-2469)ACC > TCC p.T823S TNN c.782A > T c.(781-783)CAG > CTG p.Q261L TNN c.2392G > T c.(2392-2394)GAC > TAC p.D798Y TNN c.3711C > A c.(3709-3711)TGC > TGA p.C1237* TNN c.3028C > A c.(3028-3030)CCA > ACA p.P1010T TNN c.212C > T c.(211-213)TCG > TTG p.S71L TP53 c.992A > G c.(991-993)CAG > CGG p.Q331R TP53 c.403_403delT c.(403-405)TGCfs p.C135fs TP53 c.853G > A c.(403-405)TGCfs pE285K TP53 c.820G > T c.(820-822)GTT > TTT p.V274F TP53 c.659A > G c.(658-660)TAT > TGT p.Y220C TP53 c.586C > T c.(586-588)CGA > TGA p.R196* TP53 c.476C > T c.(475-477)GCC > GTC p.A159V TP53 c.724T > C c.(724-726)TGC > CGC p.C242R TP53 c.820G > T c.(820-822)GTT > TTT p.V274F TP53 c.659A > G c.(658-660)TAT > TGT p.Y220C TP53 c.749C > T c.(748-750)CCC > CTC p.P250L TP53 c.839G > A c.(838-840)AGA > AAA p.R280K TP53 c.742C > T c.(742-744)CGG > TGG p.R248W TP53 c.527G > A c.(526-528)TGC > TAC p.C176Y TP53 c.919_splice c.e8 + 1 p.A307_splice TP53 c.672_splice c.e6 + 1 p.E224_splice TP53 c.783_splice c.e8 − 1 p.S261_splice TP53 c.738G > C c.(736-738)ATG > ATC p.M246I TP53 c.473G > T c.(472-474)CGC > CTC p.R158L TP53 c.414_414delC c.(412-414)GCCfs p.A138fs TP53 c.578A > G c.(577-579)CAT > CGT p.H193R TP53 c.490A > T c.(490-492)AAG > TAG p.K164* TP53 c.524G > A c.(523-525)CGC > CAC p.R175H TP53 c.743G > T c.(742-744)CGG > CTG p.R248L TP53 c.818G > T c.(817-819)CGT > CTT p.R273L TP53 c.476C > A c.(475-477)GCC > GAC p.A159D TP53 c.830G > T c.(829-831)TGT > TTT p.C277F TP53 c.742C > T c.(742-744)CGG > TGG p.R248W PEX5L c.535G > A c.(535-537)GAT > AAT p.D179N PEX5L c.686C > A c.(685-687)TCT > TAT p.S229Y PEX5L c.11G > A c.(10-12)GGA > GAA p.G4E PEX5L c.266_267CC > AA c.(265-267)ACC > AAA p.T89K PEX5L c.1063C < G c.(1063-1065)CAG > GAG p.Q355E PEX5L c.115G > A c.(115-117)GAT > AAT p.D39N PEX5L c.1713G > T c.(1711-1713)TTG > TTT p.L571F PEX5L c.337G > A c.(337-339)GAC > AAC p.D113N

While the invention has been particularly shown and described with reference to specific embodiments (some of which are preferred embodiments), it should be understood by those having skill in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the present invention as disclosed herein. 

What is claimed is: 1) A method for treating a patient having cancer, comprising: obtaining a sample derived from the patient; determining that the sample does not have a mutation in TP53 gene which results in Q331R, C135fs, E285K, V274F, Y220C, P250L, R175H, R248Q, R280K, R248L, C176Y, A307_splice, R273L, R158L, A138fs, H193R, A159D, C277F, R248W, Y220C, V274F, R196*, E224_splice, K164*, M246I, A159V, S241F, C242R, S261_splice, E339* in TP53 protein; and administering an MEK inhibitor to the patient. 2) The method of claim 1, wherein the sample is a cancer cell or tissue derived from the patient. 3) The method of claim 1, wherein the MEK inhibitor is Trametinib. 4) The method of claim 1, wherein the determining step comprises amplifying at least a portion of the TP53 gene with an oligonucleotide as primer to generate an amplification product. 5) The method of claim 1, wherein the determining step comprises contacting the sample with an oligonucleotide which specifically hybridizes to the mutation of the TP53 gene to form a complex. 6) The method of claim 1, wherein the determining step involves an assay selected from the group consisting of sequencing, polymerase chain reaction (PCR), mass-spectrometric genotyping, HPLC, SSPC and a hybridization-based assay. 7) The method of claim 1, wherein the determining step further comprises determining if the patient has a second mutation in one or more genes selected from the group consisting of ADAM12, COL14A1 and TNN. 8) The method of claim 7, wherein the mutation in ADAM12 gene results in Q650K, R240L, C440Y, Q228E, H247D, M322I, T97fs, P168L or G308E in ADAM12 protein; wherein the mutation in COL14A1 gene results in R178W, L713 splice, Q1272K, L4791, L1295F, E1024K, P1467S, G737R, K1023T, G966C, S1512fs in COL14A1 protein; wherein the mutation in TNN gene results in V353M, Y296S, A733P, D707Y, D471Y, P1010T, S71L, D457Y, P1155L, R476C, Q872H, Q261L, D798Y, C1237*, D67N and T823S in TNN protein. 9) A method for treating a patient having cancer, comprising: obtaining a sample derived from the patient; determining that the sample has a mutation in TP53 gene which results in Q331R, C135fs, E285K, V274F, Y220C, P250L, R175H, R248Q, R280K, R248L, C176Y, A307_splice, R273L, R158L, A138fs, H193R, A159D, C277F, R248W, Y220C, V274F, R196*, E224_splice, K164*, M246I, A159V, S241F, C242R, S261_splice, E339* in TP53 protein; and administering a therapeutic agent to the patient, wherein the therapeutic agent is selected from the group consisting of a c-Met inhibitor, an agent targeting PI3K-Akt-mTOR signaling pathway, a chemotherapeutic agent, an anti-metabolite, an anti-hormonal agent, and an angiogenesis inhibitor. 10) The method of claim 9, wherein the sample is a cancer cell or tissue derived from the patient. 11) The method of claim 9, wherein the determining step comprises amplifying at least a portion of the TP53 gene with an oligonucleotide as primer to generate an amplification product. 12) The method of claim 9, wherein the determining step comprises contacting the sample with an oligonucleotide which specifically hybridizes to the mutation of the TP53 gene to form a complex. 13) The method of claim 9, wherein the determining step involves an assay selected from the group consisting of sequencing, polymerase chain reaction (PCR), mass-spectrometric genotyping, HPLC, SSPC and a hybridization-based assay. 14) The method of claim 9, wherein the determining step further comprises determining if the patient has a second mutation in one or more genes selected from the group consisting of ADAM12, COL14A1 and TNN. 15) The method of claim 14, wherein the mutation in ADAM12 gene results in Q650K, R240L, C440Y, Q228E, H247D, M322I, T97fs, P168L or G308E in ADAM12 protein; wherein the mutation in COL14A1 gene results in R178W, L713 splice, Q1272K, L4791, L1295F, E1024K, P1467S, G737R, K1023T, G966C, S1512fs in COL14A1 protein; wherein the mutation in TNN gene results in V353M, Y296S, A733P, D707Y, D471Y, P1010T, S71L, D457Y, P1155L, R476C, Q872H, Q261L, D798Y, C1237*, D67N and T823S in TNN protein. 